JP5670181B2 - Methods for preparing active ingredients in hot spring water and compositions containing them - Google Patents

Description

  The present invention relates to a novel method for culturing microorganisms that imparts improved properties to microorganisms, particularly for use in the cosmetic or pharmaceutical field. The invention also relates to the use of the bacteria obtained by these methods or extracts thereof, in particular in the preparation of compositions having cosmetic or skin use.

  The present invention relates to non-photosynthetic and non-growth filamentous fungi (especially Beggiatoales) defined according to the classification of Bergey's Manual of Systematic Bacteriology (vol. 3, sections 22 and 23, 9th edition, 1989). ), And in particular culture of bacteria belonging to the genera Beggiatoa, Vitreoscilla, Flexithrix or Leucothrix).

  For the culture of such Beggiatoaceae bacteria, the culture by growth in a fermentor in a gently agitated medium is described in application FR2283223.

  Application WO94 / 02158 teaches the preparation of immunomodulatory compositions from the envelopes of bacteria of the genus Vegia toa or Vitreostilla. These bacteria are cultivated in a medium prepared with distilled water and monosaccharides, in particular glucose, as a carbon source.

  Furthermore, the filamentous fungi, or fractions thus obtained show the advantageous properties described, for example, against the signs of aging in EP681831 or to treat sensitive skin in EP761204.

FR2283223 WO94 / 02158 EP681831 EP761204 EP641557

Bergey's Manual of Systematic Bacteriology (vol. 3, sections 22 and 23, 9th edition, 1989) Heller, Ann. Sci. Nat. Biol. Veg., 14, 1-223 (1953) Noris and Ribbons, Methods in Microbiology, Vol. 5B, Academic Press (1971)

  There remains a need to find improved methods of producing such microorganisms.

  In connection with the present invention, it is possible to obtain a bacterial mass exhibiting improved properties by replacing all or part of the distilled water conventionally present in the medium for culturing filamentous fungi with mineral water Was found to be.

  The subject of the present invention is therefore a method for the preparation of an active ingredient for cosmetics or skin, comprising at least one non-photosynthetic and non-growth filamentous fungus, preferably at least one sulfur-free A method comprising culturing in a medium containing mineral spring water and / or hot spring water.

  Sulfite water generally contains bacteria with autotrophic metabolism that play a role in the sulfur cycle. The sulfur-free water according to the present invention is substantially free of such sulfite reducing bacteria.

  As mentioned above, non-photosynthetic filamentous fungi include, in particular, bacteria belonging to the order of Vegiatoa, more particularly bacteria belonging to the genus Begiatoa, Vitreostilla, Flexix or Leukotrix.

  The method according to the invention is particularly suitable for the cultivation of bacteria belonging to the genus Vitreostila, more particularly bacteria of the species Vitreoscilla filiformis.

Bacteria defined as above, some already described, generally live in water and can be found especially in seawater or hot spring water. Among the bacteria that can be used, mention may be made, for example, of:
Vitreostilla Filformis (ATCC 15551)
Vitreoscilla beggiatoides (ATCC 43181)
Beggiatoa alba (ATCC 33555)
Flexithrix dorotheae (ATCC 23163)
Leucothrix mucor (ATCC 25107)
Sphaerotilus natans (ATCC 13338).

  Preferably, the method according to the invention is applied to the cultivation of bacteria corresponding to the strain deposited with the ATCC under number 15551.

  The term “hot spring water” is understood to mean hot or cold water used for its therapeutic capacity or bathing use. In the present invention, hot spring water or mineral water can be used. In general, mineral water is suitable for ingestion, but not necessarily in the case of hot spring water. Each of these waters contains, inter alia, dissolved minerals and / or trace elements. These waters are known to be used for specific therapeutic purposes due to the specific trace elements and minerals present therein.

  Preferably, in the process according to the invention, those made of hot spring water and / or mineral water showing a total mineral content of 400 mg / l or more are used.

  In the present invention, the term “total mineral content” is understood to mean the total concentration of anions and cations present in hot spring water or mineral water. In the hot spring water or mineral spring water used according to the invention, the total mineral content is generally between 400 and 900 mg / l.

  The hot spring water and / or mineral water used according to the present invention has a total mineral content of at least 700 mg / l, in particular a total concentration of carbonate and bicarbonate of at least 150 mg / l, more preferably at least 360 mg / l, and In particular, it may have a total concentration of sodium carbonate and sodium bicarbonate of more than 2 mg / l. The concentration of silicon oxide in the water used in the composition according to the invention may preferably be at least 6 mg / l, more preferably at least 9 mg / l.

  Hot spring water or mineral spring water used in the present invention is, for example, water from Vittel, water from Vichy Basin, water from Uriage, water from La Roche-Posay, water from La Bourboule, Enghien-les-Bains Water from Saint Gervais-les-Bains, water from Neris-les-Bains, water from Allevard-les-Bains, water from Digne, water from Maizires, water from Neyrac-les-Bains Water from Lons-le-Saunier, water from Eaux-Bonnes, water from Rochefort, water from Saint Christau, water from Les Fumades, and water from Tercis-les-Bains.

  Sulfur-free water, for example, water from Vittel, water from the Vichy basin, excluding sulfite, water from La Roche-Posay, water from La Bourboule, water from Enghien-les-Bains, Neris -les-Bains water, Digne water, Maizieres water, Neyrac-les-Bains water, Lons-le-Saunier water, Rochefort water, Saint Christau water, and Tercis- Selected from water from les-Bains.

  Among these waters, water with a total concentration of carbonate or bicarbonate exceeding 360 mg / l is water from Vittel, water from La Bourboule, water from Les Fumades, water from Enghien-les-Bains. Water from La Roche-Posay, Vichy basin and Uriage.

  Among these waters, water with a carbonate or bicarbonate concentration of 150 mg / l to 360 mg / l is water from Digne, water from Maizires, water from Rochefort or from Saint Gervais-les-Bains. Of water.

  Among these waters, water containing at least 2 mg / l sodium carbonate or bicarbonate is water from La Roche-Posay, water from Vittel, water from Vichy Basin or water from Uriage.

  Water containing at least 9 mg / l silicon oxide is water from La Roche-Posay, water from Vittel, water from Vichy Basin or water from Uriage.

  Hot spring water or mineral spring water that is particularly suitable for the practice of the present invention has a calcium ion concentration of 100 mg / l or more, in fact 140 mg / l.

  According to an advantageous embodiment, the hot spring water or the mineral spring water has a bicarbonate ion concentration of 300 mg / l or more. The bicarbonate, also known as bicarbonate, is present in particular at a concentration of 350 mg / l or more.

  According to an advantageous embodiment, the bacteria are cultivated in a medium comprising at least one hot spring water. The latter are especially water from Vittel, water from the Vichy Basin, water from Uriage, water from La Roche-Posay, water from La Bourboule, or water from Enghien-les-Bains, especially water from Vittel, It is possible to select from water from Vichy basin excluding sulfite water and water from La Roche-Posay.

  The water with which certain advantageous results are obtained according to the invention is selected in particular from water from La Roche-Posay and water from Vittel or water having a similar composition.

  Water from La Roche-Posay is extracted from a spring of the same name and contains bicarbonate, calcium, silicate and selenium. Water from La Roche-Posay generally contains approximately 387 mg / l bicarbonate ion, approximately 140 mg / l calcium ion and at least 40 mg / l sulfate.

  The water from Vittel is rich in calcium and mineral salts (841 mg / l), especially including 202 mg / l calcium, 402 mg / l bicarbonate and 336 mg / l sulfate.

  Culturing can be performed in particular in the following media:

Composition Concentration Autolysis Yeast Extract 0.5-5g / l
Peptone 0.5-5g / l
Anhydrous glucose 0.5-7g / l
Heller trace element 0.5-5ml / l
CaCl ₂・ 10H ₂ O 0.010〜0.200g / l

  The composition is made up to 1000 ml with distilled water and / or mineral water or hot spring water.

  Among the peptones that can be used, mention may be made, for example, of soy papain peptone.

  This medium is distinguished from the commonly used medium without catalase and sulfide. Heller trace elements are described by Heller in Ann. Sci. Nat. Biol. Veg., 14, 1-223 (1953). They are a mixture of various mineral elements recommended by Heller, not for the cultivation of bacteria, but for the nutrition of plant tissues cultured in vitro.

  Culturing can be carried out at a suitable temperature suitable for the bacterial species to be cultured. In general, this temperature is 18-40 ° C. depending on the strain. The pH of the culture medium is preferably 5.5-8.

The composition of Heller trace elements per liter of water is as follows:
ZnSO ₄・ 7H ₂ O 1g
MnSO ₄・ H ₂ O 0.076g
CuSO ₄・ 5H ₂ O 0.003g
KI 0.010g
H ₃ BO ₃ 1g
AlCl ₃・ 6H ₂ O 0.050g
NiCl ₂・ 6H ₂ O 0.030g

  The hot spring water or mineral water can replace all or part of the aqueous phase of the culture medium. They can therefore be found as a mixture in any proportion with water, in particular distilled or osmotic water present in the culture medium.

  After all the elements of the medium are mixed, the culture medium containing hot spring water and / or mineral water is conveniently sterilized. This step is carried out by methods known to those skilled in the art, such as filtration or sterilization by heating.

  Subsequently, the culture medium is inoculated with bacteria.

  Accordingly, another subject of the invention is a method of culturing microorganisms or a method of preparing cosmetic or dermatological active ingredients as defined above, wherein the culture medium comprises (i) osmotic or distilled water and ( ii) A process characterized in that it comprises a mixture of hot spring water in a ratio of (i) / (ii) 1-100, in particular 1-50, in particular 1-25.

  Surprisingly, in the context of the present invention, by including a relatively small proportion of hot spring water and / or mineral spring water in the culture medium, the microorganism contains exclusively water that is contained in distilled water and / or osmotic treatment. It has been found that advantageous properties different from those of microorganisms cultured in a medium that is water can be imparted.

  Therefore, in a medium suitable for the practice of the present invention, the amount of water introduced for the preparation of the medium in hot spring water or mineral spring water preferably corresponds to at least 0.1%, in particular 0.1-99.9%. Good results are obtained for osmotic and / or distilled water at approximately 1% or 2%, for example 0.5-20%, indeed 0.5-50% hot spring water concentrations, but these concentrations are inconvenient It can be increased without.

  In a known manner, the method comprises at least one stage in which the bacteria are recovered at the end of the culture, in particular by separating the cells from the culture medium.

  After culturing the bacteria, the biomass can be obtained by various known methods such as filtration, coagulation with alcohol (ethanol, isopropanol, isobutanol), and drying in a cylinder with a scrape pre-layer (starch, diatom, etc.). Or can be isolated by lyophilization. Preconcentration, for example at 80 ° C. under reduced pressure, improves this separation.

  The operation of rupturing the envelope can be performed, for example, by the action of ultrasonic waves. In addition, extracts can be prepared using alcohols such as ethanol or propanol.

  The lipopolysaccharide extract can also be prepared according to a known method. See, for example, Noris and Ribbons, Methods in Microbiology, Vol. 5B, Academic Press (1971). A commonly used method is the well-known “Westphal” method (or related method), which is to perform extraction with a phenol / water mixture at 65 ° C. The extract is subsequently subjected to dialysis to remove phenol.

  The present invention provides a method for preparing an active ingredient for cosmetics or skin, as defined above, comprising: (i) at least one bacterium belonging to the order of Vegia toa, a monosaccharide as a main carbon source and at least It particularly relates to a method comprising culturing in a medium containing one mineral spring water or hot spring water, and then (ii) separating the bacteria from the culture medium after fermentation and recovering the bacterial mass.

  Bacteria recovered at the end of the fermentation stage can be subjected in particular to stabilization and / or extraction treatment. This is an extract of the filamentous fungus thus obtained that can be used generally in or for the preparation of cosmetic or dermatological compositions. In a manner known per se, the extract can therefore be sterilized, in particular by filtration or autoclaving.

  The term “non-photosynthetic filamentous fungal extract” likewise means the supernatant from the culture of the bacteria, the biomass obtained after the cultivation of the bacteria, or the extract of the biomass obtained by processing of the biomass. Is understood.

  In order to prepare the extract according to the invention, the bacteria can be cultured by the method according to the invention and then separated from the resulting biomass, for example by filtration, centrifugation, coagulation and / or lyophilization.

  Therefore, after culturing, the bacteria are concentrated by centrifugation. The obtained biomass is autoclaved. This biomass can be lyophilized to constitute what is referred to as a lyophilized extract. This extract can be prepared using any lyophilization method known to those skilled in the art.

  The supernatant fraction from the biomass can be filtered into a sterile container to remove suspended particles. This supernatant fraction may be gently poured into a sterile container under aseptic conditions. According to a particular embodiment of the invention, the supernatant fraction thus obtained is used as an active ingredient for cosmetics or skin.

  Surprisingly, in the context of the present invention, the cultivation of bacteria in a medium comprising at least one hot spring water or mineral water, in particular comprising 1-100% of the water present in the culture medium, is subject to various fermentations and Specific characteristics that differ from those of the same bacteria cultured in a medium that is not supplemented with spring and / or mineral water but contains only osmotic or distilled water, at the end of the isolation phase Has been found to confer.

  According to another aspect of the present invention, replacement of all or part of the medium water for culturing bacteria with mineral water or hot spring water can increase the growth rate of bacteria and thus improve yield.

  Production can be increased by at least 10%, especially with controlled growth rates and identical and controlled culture conditions.

  Accordingly, another subject of the invention is the activity of at least one spa or mineral water, preferably sulfur-free, of Vitreostila spp. Or use as a drug to improve growth.

  The present invention also relates to a method for improving the growth and / or characteristics of a bacteria of the genus Begiatora or Vitreostila, wherein the spring water and / or mineral water is added to the culture medium in at least one culture stage. Also related.

  As mentioned above, the hot spring water or mineral water is preferably sulfur-free water and is specifically selected from water from Vittel, water from the Vichy basin excluding sulfite water and water from La Roche-Posay.

  Another subject of the present invention is a culture medium suitable for the cultivation of non-photosynthetic filamentous fungi, as defined above, wherein the water present in said medium is at least 1%, in particular 1-99.9%. Preferably 1-25% mineral spring water and / or hot spring water.

  Another subject of the invention is also the bacteria obtained by the method according to the invention, in particular the Vitreostila filiformis, and extracts thereof, and their use in compositions, wherein the bacteria, extracts or compositions are skin And / or used to improve the condition of body surface growth.

  Another subject of the invention is therefore a composition, in particular a cosmetic or dermatological composition, in a physiologically acceptable medium, at least one bacterium of the genus Vitreostilla obtained by the above method, or the latter It is a composition characterized by including an extract.

  The compositions used according to the invention can be provided in all forms suitable for applications considered in the cosmetic and dermatological field, in particular oral or topical applications.

  The term “topical” is preferably understood to mean use on the skin or body surface growth by topical application. The term “skin” also includes the scalp or mucosa unless otherwise indicated.

  The composition according to the invention can therefore be applied to any skin area, hair, nails or mucous membranes of the body and can be given in any suitable formulation form by a person skilled in the art. Such compositions are in particular aqueous or oily solutions or suspensions, oil-in-water or water-in-oil or multiple emulsions, silicone emulsions, microemulsions or nanoemulsions, aqueous or oily gels, or liquid, pasty or solid anhydrous It can be provided in the form of a product.

  For topical application to the skin, the composition may in particular be prepared by an aqueous or oily solution or dispersion of lotion or serotype, dispersion of the fatty phase in the aqueous phase (O / W) or vice versa (W / O). The resulting emulsion with a milk-type liquid or semi-liquid consistency, or an aqueous or anhydrous cream or gel-type soft consistency, else a suspension or emulsion with microcapsules or microparticles, or ions and / or It can have the form of a non-ionic vesicle dispersion. The composition can also be present in the form of a patch or controlled release formulation. These compositions are prepared according to the usual methods.

  They may be used on the hair in the form of an aqueous, alcoholic, or aqueous / alcoholic solution, or in the form of a cream, gel, emulsion or foam, or in the form of an aerosol composition that also includes a pressurized propellant.

  The composition can be provided in the form of drops for the eye and in the form of capsules, granules, gels, chewing pastes, syrups or tablets for ingestion.

  The amounts of the various constituents of the composition according to the invention are those conventionally used in the field under consideration.

  These compositions are especially creams for washing, protecting, treating or caring for the face, hands, feet, mainly anatomical folds, or body (e.g. day cream, night cream, makeup) (Removal cream, foundation cream, or sun cream), masks placed on the skin or hair, liquid foundation, make-up removal milk, body milk for protection or care, sun milk, lotion, for skin care Gels or foams such as cleansing lotions, sun lotions, artificial tanning lotions, bathing compositions, deodorizing compositions containing bactericides, aftershave gels or lotions, hair removal creams, compositions to accommodate insect stings or bites Product, analgesic composition, or certain skin diseases such as dampness Constitutes rosacea, psoriasis, a composition for treating pruritus of lichen or severe.

  The composition according to the invention can also consist of a solid preparation constituting a cleansing soap or bar.

  The composition can also be packed in the form of an aerosol composition that also includes a pressurized propellant.

  The composition according to the invention comprises a hair care composition, in particular a shampoo, a hair set lotion, a treatment lotion, a styling cream or gel, a dye composition (especially an oxidative dye composition), optionally in the form of a shampoo dye, It may be a structuring lotion, a permanent wave composition (particularly a composition for the first step of a permanent wave), a lotion or gel for combating hair loss, an antiparasitic shampoo, and the like.

  The composition may be for oral use, for example toothpaste. In this case, the composition comprises adjuvants and additives that are conventional to compositions for oral use, in particular surfactants, thickeners, humectants, abrasives such as silica, various active ingredients such as fluoride, In particular, sodium fluoride and optionally a sweetening agent such as sodium saccharin may be included.

  When the composition is an emulsion, the proportion of the fatty phase can range from 5% to 80% by weight, preferably from 5% to 50% by weight relative to the total weight of the composition. The oils, waxes, emulsifiers and coemulsifiers used in the composition in emulsion form are selected from those conventionally used in the cosmetic field. The emulsifier and coemulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, preferably from 0.5% to 20% by weight, relative to the total weight of the composition. In addition, the emulsion may contain lipid vesicles.

  When the composition is an oily solution or gel, the fatty phase can represent more than 90% of the total weight of the composition.

  In a known manner, the cosmetic composition comprises conventional adjuvants in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillings. Agents, screening agents, odor absorbers and colorants may be included. The amounts of these various adjuvants are those conventionally used in the cosmetic field, for example from 0.01% to 10% of the total weight of the composition. Depending on the nature, these adjuvants may be introduced into the fatty phase, the aqueous phase and / or the lipid globules.

  Oils or waxes that can be used in the present invention include mineral oil (fluid petrolatum), vegetable oil (liquid fraction of shea butter, sunflower oil), animal oil (perhydrosqualene), synthetic oil (pure serine oil), silicone oil or wax ( Mention may be made of cyclomethicone) and fluorinated oils (perfluoropolyether), beeswax, carnauba wax or paraffin wax. Fatty alcohols and fatty acids (stearic acid) can be added to these oils.

  As emulsifiers that can be used in the present invention, mention may be made, for example, of PEG-6 / PEG-32 / glycol stearate mixtures sold by Gattefosse under the name glyceryl stearate, polysorbate 60 and Tefose® 63. .

  As solvents that can be used in the present invention, mention may be made of lower alcohols, in particular ethanol and isopropanol, or propylene glycol.

  Hydrophilic gelling agents that can be used in the present invention include carboxyvinyl polymers (carbomers), acrylic copolymers such as acrylate / alkyl acrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums and clays. Examples of lipophilic gelling agents that can be used in the present invention include modified clays such as Bentones®, metal salts of fatty acids such as aluminum stearate and hydrophobic silica, ethylcellulose or polyethylene. Can do.

  In an advantageous embodiment, the composition according to the invention is an oil-in-water (O / W) composition, in particular a composition comprising a lipophilic active ingredient. Such a composition is based in particular on an oil sphere with a lamellar liquid crystal coating, as described in application EP 641557. Cosmetic or dermatological compositions are composed of oil-in-water emulsions formed from oil spheres with a lamellar liquid crystal coating dispersed in an aqueous phase, each oil sphere being cosmetic or dermatological And at least one lipophilic surfactant, at least one lipophilic surfactant, at least one hydrophilic surfactant and at least one fatty acid and each coated with a monolamellar or oligolamellar layer. The coated oil spheres have an average diameter of less than 500 nanometers and the aqueous phase contains a basic agent in the dissolved state.

  When the composition according to the invention is used for the cosmetic treatment of keratin substances, the active ingredients present in the oily phase are, for example, melanin regulators, lipomodulators, antiseborrheic agents, anti-aging agents, UV radiation. It is selected from drugs to counteract, keratolytic agents, antibacterial agents, antifungal agents, anti-dandruff agents, agents against hair loss, hair dyes, hair bleaches or conditioners.

Examples of active ingredients for skin and / or hair treatment that can be used in connection with the present invention may include the following compounds: D-α-tocopherol, DL-α-tocopherol, D-α -Tocopheryl acetate, DL-α-tocopheryl acetate, ascorbyl palmitate, vitamin F glycerides, vitamin Ds, especially vitamin D ₂ , vitamin D ₃ , retinol, retinyl ester, retinyl palmitate, retinyl propionate, β-carotene, D-panthenol, farnesol, farnesyl acetate, jojoba oil and blackcurrant oil rich in essential fatty acids, alkyl esters of α-hydroxy acids such as 5- (n-octanoyl) salicylic acid, salicylic acid, citric acid, lactic acid and glycolic acid, asiatic acid , Madecassoic acid, Asiaticoside, Centella asiatica total extract, β-glycyl Extracts of phospholipids, ethoxyquin, rosemary of marine origin rich in retinoic acid, α-bisabolol, ceramide such as 2-oleoylamino-1,3-octadecane, phytantriol, milk sphingomyelin, polyunsaturated essential fatty acids , Balsam extract, quercetin, dried microalgae extract (Algoxan Red from Algatec), octyl methoxycinnamate (ParsolMCX, Givaudan-Roure), butyl methoxydibenzoylmethane (Parsol 1789, Givaudan-Roure), octyl triazone (Uvinul T150, BASF), yellow, brown, black or red iron oxide, or titanium oxide, which may be in micrometer or nanometer form, or coated form.

  The amount of biomass or extract thereof cultivated in hot spring water present in the composition according to the invention can be adjusted by the person skilled in the art to obtain the desired activity, depending on the type of extract used. I will. When expressed, the amount in the composition is between 0.001% and 10% by weight, preferably 0.01% to 5% by weight, in particular at least 0.1% by weight, relative to the total weight of the composition.

  The present invention is also a method for improving the condition of skin and / or body surface growth, which is obtained by culturing in a medium containing hot spring water or mineral spring water as defined above. Also included is a method of applying a bacterium, one of its extracts, or a composition comprising them to skin and / or body surface growth.

  According to one aspect of the present invention, the present invention provides a method for the preparation of a composition, in particular a cosmetic or dermatological composition, which is obtained by culturing in a medium containing hot spring water or mineral spring water as defined above. Relates to a method of mixing at least one bacterium, or one of its extracts, with at least one physiologically acceptable medium and adjuvant suitable for the method of administration.

  The invention is illustrated in detail by the following examples.

Preparation of biomass Preparation of culture medium:
composition:
* Yeast extract 2g
* Soybean papain peptone 2g
* Glucose 2g
* Heller trace element 2ml
* CaCl ₂・ 2H ₂ O 66.21mg
* Appropriate amount for 1 liter of hot spring water from La Roche-Posay

The pH of the medium is adjusted to 5.00 with 1 molar H ₃ PO ₄ solution. The medium is sterilized at 121 ° C. for 30 minutes in a sterile autoclave. After cooling to ambient temperature, the pH is readjusted to 7.20 by addition of 1 molar KOH solution.

culture:
After inoculating the medium with Vitreostilla filiformis deposited with the ATCC under number 15551 at 1%, the culture is placed under orbital shaking at 26 ° C. and 100 rpm. After 48 hours of growth, the culture is centrifuged at 8000 g for 15 minutes. The pellets are collected and then autoclaved at 121 ° C. for 30 minutes. This biomass can be used for evaluation tests.

Comparison of the effects of biomass obtained with or without hot spring water The purpose of this study was to obtain biomass obtained by culturing in a culture medium reconstituted with osmotic water (recorded as `` biomass '' in the table) ), Biomass obtained by culturing in a medium reconstituted with hot spring water from La Roche-Posay (`` biomass LRP ''), and hot spring water from La Roche-Posay (`` hot spring water LRP '') In an attempt to determine the effect on differential gene expression in human keratinocyte cultures.

  Biomass is obtained by culturing microorganisms (Vitreostilla filiformis deposited at ATCC under number 15551) in the presence or absence of hot spring water from La Roche-Posay and is introduced at a concentration of 2%.

  These results are confirmed by RT-qPCR analysis of corneodesmosine and LEP16 markers on keratinocyte cultures.

  In keratinocyte cultures, only biomass cultured in hot spring water from La Roche-Posay stimulates the expression of corneodesmosine and LEP16. Biomass cultured in osmotic water or hot spring water from La Roche-Posay does not affect the expression of these marker genes for keratinocytes. At the same time, Merck's ultrapure water used as a control has no effect on the expression of the genes studied.

Difference in expression between Vitreostilla filiformis culture in water from La Roche-Posay and Vitroostila filiformis in osmotic treated water Isolate and express proteins expressed in Vitreostilla filiformis biomass Two-dimensional electrophoresis was used as a means for visualization, cultivated in water from La Roche-Posay on the one hand and in osmotic water on the other hand.

  The method of analysis is to separate proteins as a function of their pHi in the “horizontal” first dimension (which is isoelectric focusing or IEF). This separation occurs through a gel containing an immobilized pH gradient, also known as an IPG strip. The protein is then separated as a function of its molecular weight in the second dimension of the “vertical” movement. The results show that 2D gel proteins are very abundant, the latter being distributed in the molecular weight range between 6 kDa and 100 kDa, with many proteins in the region of acidic pH values. Rapid analysis of the image shows 6 different spots, one being overexpression of two proteins in biomass grown in water from La Roche-Posay and the other in culture in osmotic water. There are four proteins that appear to be more expressed in biomass.

  These results indicate that there is a difference in the level of protein expression in biomass cultured in osmotic treated water compared to protein expression in biomass cultured in La Roche-Posay water.

Increased growth with water from Vittel
The culture test of Vitreostilla filformis in a continuous manner in a 10 liter fermenter starts with a medium partially reconstituted with osmotic water (control test) and mineral water from Vittel (2%). Carried out. The growth rate by the continuous culture method was fixed at 0.12 h ⁻¹ , and the culture conditions were the same in the two tests. Biomass was concentrated by centrifuging continuously harvested culture volumes. The growth of the culture can be monitored by measuring the absorbance at 600 nm. The obtained sludge was stabilized at 121 ° C. for 30 minutes by an autoclave. From the collected sludge solids and amount, the cell concentration of the corresponding culture can be determined (in g / l) from the centrifuged culture volume.

  Exam summary table:

result:
Compared to control cultures in media reconstituted with osmotic water, the use of water from Vittel improves bacterial growth and increases cell yield by 13%.

Composition comprising Vitreostilla filiformis extract

  Prepare phases A, B, C and D, followed by mixing:

  The composition thus obtained can be applied to the face, neck, hands and / or whole body in the morning and / or evening.

Claims (6)

  A method for the preparation of an active ingredient for cosmetics or skin, comprising at least one non-photosynthetic and non-plant-forming filamentous fungus deposited with the ATCC under the reference number ATCC 15551 in water from La Roche-Posay And culturing in a medium comprising at least one sulfur-free mineral spring water and / or hot spring water selected from water from Vittel.   (i) At least one bacterium belonging to the family Begia Toa, a strain deposited with the ATCC under the reference number ATCC 15551, with monosaccharides as the main carbon source, water from La Roche-Posay and water from Vittel Culturing in a medium containing at least one kind of mineral water or hot spring water selected from: and (ii) after fermentation, separating the bacteria from the culture medium and recovering the bacterial mass, A method for preparing an active ingredient for cosmetics or skin according to claim 1.   The method according to claim 2, characterized in that the bacterial mass recovered at the end of the fermentation stage is subjected to a stabilization treatment.   4. A culture medium comprising a mixture of (i) osmotic or distilled water and (ii) hot spring water in a ratio of (i) / (ii) 1-100. The method according to one item. Activity and / or growth of a bacterium belonging to the genus Vitreostilla, a strain deposited with the ATCC under the reference number ATCC 15551, of at least one sulfur-free hot spring water or mineral spring water as defined in any one of claims 1 to 4 Use as a drug to improve. In media that are physiologically acceptable, obtained by the method according to any one of claims 1 to 5, the reference numeral ATCC15551 is a strain deposited with the ATCC Bitoreosuchira genera least one bacterial or said, A cosmetic or dermatological composition characterized in that it comprises an extract of bacteria .